Dialkylhydroxybenzoic acid derivatives containing metal chelating groups and their therapeutic uses

ABSTRACT

The present invention provides novel dialkylhydroxybenzoic acid derivatives containing metal chelating groups and the use of the novel compounds as therapeutics for treating and/or preventing various medical dysfunctions and diseases arising from reactive oxygen species and/or excess Zn ions, in particular stroke, Parkinson&#39;s disease, Alzheimer&#39;s disease. The compounds of the invention have not only low toxicity but also similar or superior LPO inhibition activity to references. They also effectively inhibit the cerebral neuronal cell death by ROS and/or zinc ion, and show neuroprotective effects against ischemic neuronal degeneration.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to novel dialkylhydroxybenzoic acidderivatives containing metal chelating groups, and pharmaceuticalcompositions containing the novel compounds as active ingredients, moreparticularly, to novel dialkylhydroxybenzoic acid derivatives containingmetal chelating groups, and to the use of the novel compounds astherapeutics for treating and/or preventing various medical dysfunctionsand diseases caused by reactive oxygen species and/or excess Zn ions, inparticular stroke, Parkinson's disease, and Alzheimer's disease.

[0003] 2. Description of the Prior Art

[0004] According to Harman's free-radical theory of ageing, successiveoxidation attacks create “oxidative stress” conditions, that is, createan imbalance between the protective systems in favour of thepro-oxidants. Such attacks result in numerous molecular modifications,especially of polyunsaturated membrane lipids, proteins and nucleicacids. Human and animal organisms possess various defense mechanismsthat act in synergy. Those mechanisms are of an enzymatic nature(superoxide dismutase, catalase, and glutathione peroxidase) or of anon-enzymatic nature (such as vitamins E and C, which enablephysiological control of free-radical activity). With ageing, however,that protection becomes less efficient, not to say inefficient,especially as a result of the decreased activity of a large number ofenzymes including those involved in such defense mechanisms.Consequently, for some disorders associated with ageing, such asatherosclerosis, cataract, non-insulin-dependent diabetes, cancer orchronic neurodegenerative disorders, numerous studies have been able todemonstrate that such conditions are associated with those “oxidativestress” conditions.

[0005] The central nervous system is especially sensitive to “oxidativestress” because of its high oxygen consumption, the relatively lowlevels of its antioxidant defenses and the high iron concentration ofsome cerebral regions. This explains why “oxidative stress” might be oneof the main etiological factors of cerebral ageing, as well as of acutecentral nervous system disorder such as stroke, neurodegenerativedisorders such as Parkinson's disease, Alzheimer's disease, andneurodegeneracies of the basal ganglia. The rate of occurrence ofneurodegenerative disorders of central nervous system increasesworldwide. Stroke occupies the third highest cause of death followingcardiovascular diseases and malignant tumors (see: Parnetti, L. et al.,Drug, 53:752 (1997)).

[0006] Antioxidants protecting neuron cell of brain from oxidativestress include vitamin E derivatives such as Trolox (see: J. Med. Chem.,38:453 (1995)), glutathione peroxidase (hereinafter, referred to as“GPx”) mimics (see: Daiichi Pharmaceutical Co., Ltd., Annual Report(1999); WO 9808831; U.S. Pat. No. 5,008,394; J. Am. Chem. Soc.,119:2079-2083 (1997); Adv. Pharmacol., 38:229 (1996)), superoxidedismutase (SOD) mimics (see: U.S. Pat. No. 5,827,880), and spin trappingagents (see: J. Med. Chem., 39:4988 (1996); U.S. Pat. No. 5,475,032).

[0007] A GPx mimic is synthesized compound mimicking the function ofselenocystein from GPx active site. A well-known GPx mimic, Ebselenseems to have no major toxicity in preclinical and clinical tests and itis proposed as a potential drug for stroke. Ebselen is, however, verylittle soluble in water, even in the presence of an excess ofglutathione (GSH), which limits its pharmacological applications.

[0008] Spin trapping agents may be developed as an antioxidant if theycan trap hazardous free radicals enough, which includeα-phenyl-N-tert-butylnitrone (PBN), and various derivatives of PBN havebeen developed. Generally, nitrone moiety increases the solubility ofcompounds in water. However, it has revealed shortcomings such as a lowlipid peroxidation inhibition activity in vitro and a low protection ofbrain cells in vivo (see: Fevig, Thomas L. et al., J. Med. Chem.,39:4988-4996 (1996)).

[0009] On the other hand, zinc ion, which is present with highconcentration (>10 mM) in synaptic vesicle storing brainneurotransmitters, is a necessary element in normal function of humancells. As soon as a neuron is stimulated, zinc ion is released intointerstitial fluid and then plays a crucial role in signal transmissionfrom neuron to neuron, especially signal transmission by glutamate.

[0010] Furthermore, a variety of studies have reported that zinc ion inthe synapse may play a central role in the pathological phenomena ofcentral nervous system. The exposure of brain cortex neuron cells toexcess zinc results in the immediate neuron cell injury, while theconcentration of zinc is similar to that of zinc released from the brainin convulsion or ischemia. Hence, zinc is supposed to evoke neuron cellinjury, by way of the influx of excess zinc into neuron cells. Inaccordance with the above hypothesis, it was found that translocation ofsynapse zinc in neuron cells was a main cause of selective neuronal cellinjury after transient global cerebral ischemia rather thanexcitotoxicity by calcium (see: Koh, J.-Y et al, Science, 272:1013-1016(1996); Kim, Y.-H. et al, Neuroscience, 89: 175-182 (1999)). It is moreplausible that the neurotoxicity by the translocation of zinc may play acentral role in acute neuron cell death derived from focal ischemia.Therefore, the chelation of zinc is supposed to be effective onpersisting protection of neuron cells. However, the zinc chelator forthe treatment and prevention of neurodegenerative disease of centralnervous system is not developed yet.

SUMMARY OF THE INVENTION

[0011] The present inventors synthesized novel compounds by introducingmetal chelating group into a novel structure of phenolic typeantioxidant, a reactive oxygen species (ROS) scavenger, and they foundthat the said compounds possess a protective activity in cerebral neuroncells against zinc ions, reactive oxygen species (ROS) andneuro-excitotoxic factors, while showing a low toxicity. As a result,the said compounds could be potential drug candidates for the treatmentand prevention of cell death of brain cells.

[0012] The first object of the present invention is, therefore, toprovide neuroprotective novel dialkylhydroxy benzoic acid derivativescontaining metal chelating groups.

[0013] The second object of the invention is to provide pharmaceuticalcompositions comprising the said compounds as an active ingredient forthe treatment and prevention of medical dysfunctions and diseases suchas stroke, Parkinson's disease, and Alzheimer's disease caused byreactive oxygen species and/or excess Zn ions.

[0014] The third object of the invention is to provide a method fortreating a living body afflicted with a condition requiring antioxidantsand/or metal chelating agents, in particular acute and progressiveneurodegenerative disorders, by way of administering to the living bodythe said pharmaceutical preparations.

BRIEF DESCTIPTION OF DRAWINGS

[0015] The above and the other objects and features of the presentinvention will become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

[0016]FIG. 1-a is a graph showing the results of combined treatment ofTrolox and Fe²⁺ toxin.

[0017]FIG. 1-b is a graph showing the results of combined treatment ofTrolox and Zn²⁺ toxin.

[0018]FIG. 2-a is a graph showing the results of combined treatment ofcompound obtained in Example 1 and Fe²⁺ toxin.

[0019]FIG. 2-b is a graph showing the results of combined treatment ofcompound obtained in Example 1 and Zn²⁺ toxin.

[0020]FIG. 3-a is a graph showing the results of combined treatment ofcompound obtained in Example 2 and Fe²⁺ toxin.

[0021]FIG. 3-b is a graph showing the results of combined treatment ofcompound obtained in Example 2 and Zn²⁺ toxin.

[0022]FIG. 4-a is a graph showing the results of combined treatment ofcompound obtained in Example 3 and Fe²⁺ toxin.

[0023]FIG. 4-b is a graph showing the results of combined treatment ofcompound obtained in Example 3 and Zn²⁺ toxin.

[0024]FIG. 5-a is a graph showing the results of combined treatment ofcompound obtained in Example 4 and Fe²⁺ toxin.

[0025]FIG. 5-b is a graph showing the results of combined treatment ofcompound obtained in Example 4 and Zn²⁺ toxin.

[0026]FIG. 6-a is a graph showing the results of combined treatment ofcompound obtained in Example 5 and Fe²⁺ toxin.

[0027]FIG. 6-b is a graph showing the results of combined treatment ofcompound obtained in Example 5 and Zn²⁺ toxin.

[0028]FIG. 7-a is a graph showing the results of combined treatment ofcompound obtained in Example 6 and Fe²⁺ toxin.

[0029]FIG. 7-b is a graph showing the results of combined treatment ofcompound obtained in Example 6 and Zn ²⁺ toxin.

[0030]FIG. 8-a is a graph showing the results of combined treatment ofcompound obtained in Example 7 and Fe²⁺ toxin.

[0031]FIG. 8-b is a graph showing the results of combined treatment ofcompound obtained in Example 7 and Zn²⁺ toxin.

[0032]FIG. 9 is a graph showing the level of cell damage as thetreatment concentration of Trolox increases.

[0033]FIG. 10 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 1 increases.

[0034]FIG. 11 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 2 increases.

[0035]FIG. 12 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 3 increases.

[0036]FIG. 13 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 4 increases.

[0037]FIG. 14 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 5 increases.

[0038]FIG. 15 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 6 increases.

[0039]FIG. 16 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 7 increases.

[0040]FIG. 17-a is a graph showing the protection level of cell damagein case of the treatment of the compound of the invention afterischemia.

[0041]FIG. 17-b is a photomicrograph showing the protection level ofcell damage in case of the treatment of the compound of the inventionafter ischemia.

DETAILED DESCRIPTION OF THE INVENTION

[0042] In the first aspect, the present invention provides noveldialkylhydroxybenzoic acid derivatives containing metal chelating groupswith the following formula (I):

[0043] wherein,

[0044] R₁ denotes hydrogen, or a straight or branched acyl;

[0045] R₂ denotes hydrogen or alkyl; and,

[0046] R₃ denotes metal chelating groups such as CO₂H, CONH₂, CONR₄R₅(where, R₄ denotes hydrogen, alkyl or aryl; and, R₅ denotes heterocyclicunsaturated or saturated radical having 1 to 4 heteroatoms of elementsnitrogen, oxygen and/or sulfur from the group comprising furanyl,oxazolyl, isooxazolyl, thiophenyl, thiazolyl, isothiazolyl, pyrrolyl,imidazolyl, pyrazolyl, oxadiazolyl, thiadiazoyl, pyridyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, it beingpossible for the heterocyclic radical to be substituted once or twice,identically or differently, by halogen, C₁₋₂-alkyl, C₁₋₄-alkoxy,C₁₋₄-alkylthio, hydroxy, mercapto, trifluoromethyl, nitro, phenyl,nitrile, carboxy or C₁₋₄-alkoxycarbonyl).

[0047] In this context, preferred compounds include derivatives in which

[0048] R₁ represents hydrogen, straight or branched C₁₋₁₈ alkylcarbonyl;

[0049] R₂ represents hydrogen, straight or branched C₁₋₈-alkyl; and,

[0050] R₃ represents CO₂H, CONH₂, or amides CONR₄R₅ (where, R₄represents hydrogen, C₁₋₄-alkyl, aralkyl or cycloalkyl; and, R₅ denotesheterocyclic unsaturated or saturated radical having 1 to 4 heteroatomsof elements nitrogen, oxygen and/or sulfur from the group comprisingfuranyl, oxazolyl, isooxazolyl, thiophenyl, thiazolyl, isothiazolyl,pyrrolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazoyl, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl itbeing possible for the heterocyclic radical to be substituted once ortwice, identically or differently, by halogen, C₁₋₂-alkyl, C₁₋₄-alkoxy,C₁₋₄-alkylthio, hydroxy, mercapto, trifluoromethyl, nitro, phenyl,nitrile, carboxy and/or C₁₋₄-alkoxycarbonyl)

[0051] More preferred compounds include derivatives in which

[0052] R₁ represents hydrogen, acetyl, propionyl;

[0053] R₂ represents hydrogen, methyl, ethyl, propyl, butyl, isopropyl,tert-butyl, cyclopentyl, cyclohexyl; and,

[0054] R₃ represents CO₂H, CONH₂, or amides CONR₄R₅ (where, R₄represents hydrogen, methyl, ethyl, propyl, benzyl, cyclopentyl orcyclohexyl; and, R₅ denotes heterocyclic unsaturated or saturatedradical having 1 to 4 heteroatoms of elements nitrogen, oxygen and/orsulfur from the group comprising oxazolyl, thiazolyl, isothiazolyl,imidazolyl, pyrazolyl, thiadiazoyl, pyridyl, pyrimidinyl, pyrazinyl,pyridazinyl, triazinyl, triazolyl, tetrazolyl it being possible for theheterocyclic radical to be substituted once or twice, identically ordifferently, by fluorine, chlorine, bromine, methyl, methoxy, ethoxy,methylmercapto, hydroxy, mercapto, trifluoromethyl, nitro, phenyl,nitrile, carboxy, methoxycarbonyl and/or ethoxycarbonyl).

[0055] The compounds of the invention possess similar or better LPOinhibition activity to the reference compounds of Trolox and Ebselen.While showing lower toxicity, they also effectively inhibit the cerebralneuronal cell injury caused by ROS and/or zinc ion and showsneuroprotective effects against ischemic neuronal degeneration.

[0056] The compounds of the invention, particularly the compoundsynthesized in Example 4 below, have a very low toxicity LD₅₀≧6,950mg/kg in the case of oral administration in rats, and ≧800 mg/kg in thecase of intraperitoneal administration in rats. Therefore, one of theadvantages of the invention is that the novel compounds can beadministered at vastly higher levels than certain other knownantioxidants, such as Ebselen (LD₅₀ values of Ebselen obtained on micewere ≧6,810 mg/kg in the case of oral administration, and 740 mg/kg inthe case of intraperitoneal administration. Similarly, the LD₅₀ valuesof Ebselen obtained on rats were ≧6,810 mg/kg in the case of oraladministration and 580 mg/kg in the case of intraperitonealadministration). Accordingly, large doses of the subject compounds maybe administered immediately post stroke or other trauma to significantlyreduce oxidative damage in many cases.

[0057] In the second aspect, the present invention providespharmaceutical compositions comprising pharmaceutically acceptablecarrier and pharmaceutically effective amount of the compound of formula(I) above or pharmaceutically acceptable salts thereof. Aneuroprotective agent comprising the compound of the formula (I) asactive ingredient is preferably provided.

[0058] In the third aspect, the present invention provides a method fortreating a living body afflicted with a condition requiring anantioxidant and/or metal chelating agent, in particular acute andprogressive neurodegenerative disorders, comprising a step ofadministering to the living body said pharmaceutical composition.

[0059] As previously mentioned, the compounds of the present inventionhave been found to be effective in relieving various effects resultingfrom ROS and/or excess Zn ions. These compounds are useful astherapeutics for treating and/or preventing a wide variety of medicaldysfunctions and diseases including, but not limited to, acute centralnervous system (CNS) disorders and neurodegerative conditions.

[0060] The compounds of the invention, as pharmaceuticals, are typicallyadministered in the form of a pharmaceutical composition comprising atleast one active compound of the invention and a pharmaceuticallyacceptable carrier or vehicle suitable for use in pharmaceuticalcompositions.

[0061] In general, the compounds of the invention are administered in apharmaceutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof relevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like. The dosage used ranges from 10 mg to500 mg in one or several administrations per day.

[0062] The pharmaceutical compositions of the invention can beadministered by a variety of routes including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular, and intranasal. Depending onthe intended route of delivery, the compounds are preferably formulatedas either injectable or oral compositions.

[0063] The compositions for oral administration can take the form ofbulk liquid dilutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. In such compositions, dialkyl hydroxybenzoic acidderivatives containing metal chelating groups of the invention isusually a minor component (from about 0.1 to about 50% by weight orpreferably from about 1 to about 40% by weight) with the remainder beingvarious vehicles or carriers and processing acids helpful for formingthe desired dosing form.

[0064] Liquid forms suitable for oral administration may include asuitable aqueous or nonaqueous vehicle with buffers, suspending anddispensing agents, colorants, flavors and the like. Solid forms mayinclude, for example, any of the following ingredients, or compounds ofa similar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; a sweetening agent such as sucrose or saccharin; or aflavoring agent such as peppermint, methyl salicylate, or orangeflavoring.

[0065] Injectable compositions are typically based upon injectablesterile saline or phosphate-buffered saline or other injectable carriersknown in the art. As before, the present compound in such compositionsis typically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

[0066] The above described components for orally administrable orinjectable compositions are merely representative. Other materials aswell as processing techniques and the like are set forth in Part 8 ofRemington's Pharmaceutical Sciences, 17th edition, 1985, Mack PublishingCompany, Easton, Pa., which is incorporated herein by reference.

[0067] The compounds of the invention can also be administered insustained release forms or from sustained release drug delivery systems.A description of representative sustained release materials can be foundin the incorporated materials in Remington's Pharmaceutical Sciences.

[0068] The following Examples are provided to illustrate the inventionand are not to be construed in any way as limiting the scope of theinvention.

Example 1 Synthesis of3,5-Di-tert-butyl-4-hydroxy-N-pyridin-2-yl-benzamide (2)

[0069]

[0070] To a solution of 0.1 g (0.42 mmol) of compound 1 in CH₂Cl₂ (1 mL)were added 96 mg (0.50 mmol) of1-(3-dimethylamino)propyl-3-ethylcarbodimide hydrochloride (EDC), 26 mg(0.21 mmol) of 4-dimethylaminopyridine (DMAP) and 60 mg (0.63 mmol) of2-aminopyridine. After stirring for 12 hours at room temperature, H₂Oand CH₂Cl₂ were added. The organic layer was separated, dried overanhydrous MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash column chromatography (silica, Hex:EtOAc8:1) to give 53 mg of compound 2 in 70% yield.

[0071]¹H NMR (CDCl₃): δ 8.68 (s, NH), 8.39 (d, 1H, J=8.4 Hz), 8.23 (d,1H, J=2.4 Hz), 7.75 (s, 3H), 7.03 (t, 1H, J=6.9 Hz), 5.67 (s, OH), 1.47(s, 18H);

[0072]¹³C NMR (CDCl₃): δ 166.8, 157.9, 152.3, 148.1, 138.8, 136.6,125.7, 124.9, 119.9, 114.5, 34.8, 30.5

Example 2 Synthesis of 3,5-Di-tert-butyl-4-hydroxy-N-pyridin-2-yl methylbenzamide (3)

[0073]

[0074] To a solution of 0.1 g (0.42 mmol) of compound 1 in CH₂Cl₂ (1 mL)were added 96 mg (0.50 mmol) of1-(3-dimethylamino)propyl-3-ethylcarbodimide hydrochloride (EDC), 26 mg(0.21 mmol) of 4-dimethylaminopyridine and 0.07 mL (0.63 mmol) of2-aminomethylpyridine. After stirring for 3 hours at room temperature,H₂O and CH₂Cl₂ were added. The organic layer was separated, dried overanhydrous MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash column chromatography (silica,Hex:EtOAc=5:1) to give 110 mg of compound 3 in 80% yield.

[0075]¹H NMR (CDCl₃): δ 8.55 (d, 2H, J=4.8 Hz), 7.64 (s, 3H), 7.35 (d,2H, J=8.0 Hz), 7.20 (t, 1H, J=5.4 Hz), 5.55 (s, OH), 4.75 (d, 2H, J=5.1Hz), 1.46 (s, 18H);

[0076]¹³C NMR (CDCl₃): δ 168.5, 157.3, 157.2, 149.5, 137.2, 136.2,125.9, 124.66, 122.69, 45.3, 34.8, 30.5

Example 3 Synthesis of3,5-Di-tert-butyl-4-hydroxy-N-(8-hydroxyquinolin-5-yl) benzamide (4)

[0077]

[0078] To a solution of 0.1 g (0.42 mmol) of compound 1 in CH₂Cl₂ (5 mL)were added 96 mg (0.50 mmol) of 1-(3-dimethylamino)propyl-3-ethylcarbodiimide hydrochloride (EDC), 26 mg (0.21 mmol) of4-(dimethyl)-amino pyridine (DMAP), 2 mL of triethylamine and 147 mg(0.63 mmol) of 5-amino-8-hydroxyquinoline 2HCl salt. After stirring for12 hours at room temperature, H₂O and CH₂Cl₂ were added. The organiclayer was separated, dried over anhydrous MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography (silica, Hex:EtOAc=5:1) to give 64 mg of compound4 in 40% yield.

[0079]¹H NMR (CDCl₃): δ 8.88 (s, 2H), 8.25 (s, 3H), 7.36 (t, 1H, J=6.8Hz), 7.29 (d, 1H, J=9.3 Hz), 6.76 (d, 1H, J=8.1 Hz), 5.74 (s, OH), 4.18(s, OH), 1.49 (s, 18H);

[0080]¹³C NMR (CDCl₃): δ 179.0, 166.9, 159.0, 150.7, 140.8, 140.5,136.2, 130.1, 128.5, 122.4, 120.9, 120.3, 120.0, 109.5, 34.8, 30.6

Example 4 Synthesis of 3,5-Di-tert-butyl-4-hydroxy-N-thiazol-2-ylbenzamide (5)

[0081]

[0082] To a solution of 3.69 g (15.5 mmol) of compound 1 in DMF (5 mL)were added 2.9 mL (18.6 mmol) of diisopropylcarbodiimide (DIC), 2.85 g(18.6 mmol) of N-hydroxybenzotriazole (HOBT), and 2.33 g (23.3 mmol) of2-aminothiazole. After stirring for 20 hours at room temperature, H₂Oand EtOAc were added. The organic layer was separated, dried overanhydrous MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash column chromatography (silica,Hex:EtOAc=5:1) to give 2 g of compound 5 in 70% yield.

[0083]¹H NMR (CDCl₃): δ 7.83 (s, 2H), 7.05 (d, 1H, J=3.6 Hz), 6.90 (d,1H, J=3.6 Hz), 1.44 (s, 18H);

[0084]¹³C NMR (CDCl₃): δ 166.5, 160.7, 158.2, 137.4, 125.6, 123.8,113.3, 34.6, 30.2

Example 5 Synthesis of2-(3,5-Di-tert-butyl-4-hydroxy-benzoylamino)-thiazole-4-carboxylic acidethyl ester (6)

[0085]

[0086] To a solution of 0.15 g (0.63 mmol) of compound 1 in DMF (1 mL)were added 0.12 mL (0.76 mmol) of diisopropylcarbodiimide (DIC), and 112mg (0.95 mmol) of ethyl 2-aminothiazole-4-carboxylate. After stirringfor 24 hours at room temperature, H₂O and CH₂Cl₂ were added. The organiclayer was separated, dried over anhydrous MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashcolumn chromatography (silica, Hex:EtOAc=6:1) to give 74 mg of compound6 in 30% yield.

[0087]¹H NMR (CDCl₃): δ 10.43 (s, NH), 7.86 (s, 1H), 7.77 (s, 2H), 5.76(s, OH), 4.29 (q, 2H, J=11.5 Hz), 1.45 (s, 18H), 1.33 (t, 3H, J=7.1 Hz);

[0088]¹³C NMR (CDCl₃): δ 165.7, 161.5, 159.3, 158.6, 141.8, 136.7,125.2, 122.6, 122.4, 61.5, 34.6, 30.2, 14.4

Example 6 Synthesis of 4-Hydroxy-3,5-dimethyl-N-pyridin-2-yl-benzamide(8)

[0089]

[0090] To a solution of 30 mg (0.18 mmol) of compound 7 in DMF (5 mL)were added 0.17 mL (0.21 mmol) of diisopropylcarbodiimide (DIC) and 17mg (0.18 mmol) of 2-aminopyridine. After stirring for 10 hours at roomtemperature, saturated NaCl solution was added. The resulting mixturewas extracted with EtOAc and the organic layer was washed with water andsaturated NaCl solution several times. The organic layer was separated,dried over anhydrous MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash column chromatography(silica, Hex:EtOAc=2:1) to give 4.4 mg of compound 8 in 10% yield.

[0091]¹H NMR (CD₃CO₂D): δ 8.43 (d, 1H, J=8.6 Hz), 8.30 (d, 1H, J=4.7Hz), 8.06 (dd, 1H, J=7.4, 8.6 Hz), 7.74 (s, 2H), 7.30 (dd, 1H, J=6.3,6.4 Hz), 2.29 (s, 6H);

[0092]¹³C NMR (CD₃CO₂D): δ 168.66, 158.44, 152.16, 144.53, 142.90,130.18, 125.07, 120.96, 117.44, 16.42

Example 7 Synthesis of 4-Hydroxy-3,5-dimethyl-N-pyridine-2-yl methylbenzamide (9)

[0093]

[0094] To a solution of 30 mg (0.18 mmol) of compound 7 in DMF (5 mL)were added 0.17 mL (0.21 mmol) of diisopropylcarbodiimide (DIC) and 19mg (0.18 mmol) of 2-aminomethylpyridine. After stirring for 3.5 hours atroom temperature, saturated NaCl solution was added. The resultingmixture was extracted with EtOAc and the organic layer was washed withwater and saturated NaCl solution several times. The organic layer wasseparated, dried over anhydrous MgSO₄, filtered and concentrated underreduced pressure. The residue was purified by flash columnchromatography (silica, Hex:EtOAc=2:1) to give 13.8 mg of compound 9 in30% yield.

[0095]¹H NMR (DMSO-d₆): δ 8.81-8.72 (m, 2H), 8.49 (d, 1H, J 4.2 Hz),7.73 (dd, 1H, J=0.3, 7.5 Hz), 7.53 (s, 2H), 7.27 (d, 1H, J=7.8 Hz), 7.24(dd, 1H, J=5.8, 6.37 Hz);

[0096]¹³C NMR (DMSO-d₆): δ 166.31, 159.20, 156.09, 148.74, 136.62,127.76, 124.80, 123.63, 121.93, 120.78, 44.58, 16.64

[0097] Using the procedures described in Examples 1-7 above and theappropriate starting materials and reagents, the followingdialkylhydroxybenzoic acid derivatives containing metal chelating groupscould be prepared:

[0098] 3,5-Dimethyl-4-hydroxy-N-furan-2-yl-benzamide

[0099] 3,5-Di-tert-butyl-4-hydroxy-N-furan-2-yl-benzamide

[0100] 3,5-Dimethyl-4-acetoxy-N-furan-2-yl-benzamide

[0101] 3,5-Di-tert-butyl-4-acetoxy-N-furan-2-yl-benzamide

[0102] 3,5-Dimethyl-4-hydroxy-N-oxazol-2-yl-benzamide

[0103] 3,5-Di-tert-butyl-4-hydroxy-N-oxazol-2-yl-benzamide

[0104] 3,5-Dimethyl-4-hydroxy-N-isoxazol-3-yl-benzamide

[0105] 3,5-Di-tert-butyl-4-hydroxy-isoxazol-3-yl-benzamide

[0106] 3,5-Dimethyl-4-hydroxy-N-thiophen-2-yl-benzamide

[0107] 3,5-Di-tert-butyl-4-hydroxy-N-thiophen-2-yl-benzamide

[0108] 3,5-Dimethyl-4-hydroxy-N-thiazol-2-yl-benzamide

[0109] 3,5-Di-tert-butyl-4-hydroxy-N-thiazol-2-yl-benzamide

[0110] 3,5-Dimethyl-4-hydroxy-N-isothiazol-3-yl-benzamide

[0111] 3,5-Di-tert-butyl-4-hydroxy-N-isothiazol-3-yl-benzamide

[0112] 3,5-Dimethyl-4-hydroxy-N-(1H-pyrrol-2-yl)-benzamide

[0113] 3,5-Di-tert-butyl-4-hydroxy-N-(1H-pyrrol-2-yl)-benzamide

[0114] 3,5-Dimethyl-4-hydroxy-N-(1-methyl-1H-pyrrol-2-yl)-benzamide

[0115] 3,5-Di-tert-butyl-4-hydroxy-N-(1-methyl-1H-pyrrol-2-yl)-benzamide

[0116] 3,5-Dimethyl-4-hydroxy-N-(1H-imidazol-2-yl)-benzamide

[0117] 3,5-Di-tert-butyl-4-hydroxy-(1H-imidazol-2-yl)-benzamide

[0118] 3,5-Dimethyl-4-hydroxy-N-(1-methyl-1H-imidazol-2-yl)-benzamide

[0119]3,5-Di-tert-butyl-4-hydroxy-N-(1-methyl-1H-imidazol-2-yl)-benzamide

[0120] 3,5-Dimethyl-4-hydroxy-N-(2H-pyrazol-3-yl)-benzamide

[0121] 3,5-Di-tert-butyl-4-hydroxy-N-(2H-pyrazol-3-yl)-benzamide

[0122] 3,5-Dimethyl-4-hydroxy-N-(2-methyl-2H-pyrazol-3-yl)-benzamide

[0123]3,5-Di-tert-butyl-4-hydroxy-N-(2-methyl-2H-pyrazol-3-yl)-benzamide

[0124] 3,5-Dimethyl-4-hydroxy-N-[1,3,4]oxadiazol-2-yl-benzamide

[0125] 3,5-Di-tert-butyl-4-hydroxy-N-[1,3,4]oxadiazol-2-yl-benzamide

[0126] 3,5-Dimethyl-4-hydroxy-N-[1,3,4]thiadiazol-2-yl-benzamide

[0127] 3,5-Di-tert-butyl-4-hydroxy-N-[1,3,4]thiadiazol-2-yl-benzamide

[0128] 3,5-Dimethyl-4-hydroxy-N-pyridin-2-yl-benzamide

[0129] 3,5-Di-tert-butyl-4-hydroxy-N-pyridin-2-yl-benzamide

[0130] 3,5-Dimethyl-4-hydroxy-N-pyrimidin-2-yl-benzamide

[0131] 3,5-Di-tert-butyl-4-hydroxy-N-pyrimidin-2-yl-benzamide

[0132] 3,5-Dimethyl-4-hydroxy-N-pyridazin-3-yl-benzamide

[0133] 3,5-Di-tert-butyl-4-hydroxy-N-pyridazin-3-yl-benzamide

[0134] 3,5-Dimethyl-4-hydroxy-N-pyrazin-2-yl-benzamide

[0135] 3,5-Di-tert-butyl-4-hydroxy-N-pyrazin-2-yl-benzamide

[0136] 3,5-Dimethyl-4-hydroxy-N-[1,3,5]triazin-2-yl-benzamide

[0137] 3,5-Di-tert-butyl-4-hydroxy-N-[1,3,5]triazin-2-yl-benzamide

[0138] 3,5-Dimethyl-4-hydroxy-N-(2H-[1,2,4]triazol-3-yl)-benzamide

[0139] 3,5-Di-tert-butyl-4-hydroxy-N-(2H-[1,2,4]triazol-3-yl)-benzamide

[0140]3,5-Dimethyl-4-hydroxy-N-(2-methyl-2H-[1,2,4]triazol-3-yl)-benzamide

[0141]3,5-Di-tert-butyl-4-hydroxy-N-(2-methyl-2H-[1,2,4]triazol-3-yl)-benzamide

[0142] 3,5-Dimethyl-4-hydroxy-N-(1H-tetrazol-5-yl)-benzamide

[0143] 3,5-Di-tert-butyl-4-hydroxy-N-(1H-tetrazol-5-yl)-benzamide

[0144] 3,5-Dimethyl-4-hydroxy-N-(1-methyl-1H-tetrazol-5-yl)-benzamide

[0145]3,5-Di-tert-butyl-4-hydroxy-N-(1-methyl-1H-tetrazol-5-yl)-benzamide

Example 8 Inhibition of Lipid Peroxidation

[0146] The compounds of the present invention were tested forantioxidizing effect in terms of the repression of the radical chainreaction of a multilayer liposome.

[0147] The liposome was prepared as followings: 30 mg of commerciallyavailable soybean phosphatidylcholine (PC, Sigma Chemical Co., U.S.A.)was dissolved in 1 mL of ethanol, and 200 μL of the ethanol/PC solutionwas added to 10 mL of 10 mM Tris buffer including 50 mM NaCl (pH 7.0)with stirring.

[0148] The ability of a compound to inhibit oxidation of the liposomewas evaluated as followings: To 400 μL of the liposomes were added thetest compound (in buffer or ethanol) and histidine-FeCl₃ (167:33 μMfinal). Oxidation was initiated by the addition of FeCl₂ (33 μM finalprepared in nitrogen purged water). The mixtures was shaken at 37° C.for 15 minutes. Thereafter, tubes were treated with 1 mL of 0.67%thiobabituric acid (TBA):10% trichloroacetic acid (2:1, v/v) in 0.25 NHCl solution, containing 1.5% (v/v) t-butylhydroxytoluene (BHT) toterminate oxidation. The aliquots were heated to 100° C. for 20 minutes.After ice cooling, 1 mL of chloroform was added to 1 mL supernatant fromtubes and tubes were centrifuged. The absorbances of the resultingsupernatant were measured at 532 nm (see: Table below) InhibitorConcentration (IC₅₀) Example 1 11.88 μM Example 2 12.65 μM Example 37.79 μM Example 4 9.69 μM Example 5 11.10 μM Example 6 121.50 μM Example7 299.5 μM Ebselen 68.86 μM Trolox 10.15 μM

[0149] It can be seen from Table that the compounds of the presentinvention, especially compounds obtained in Example 1-5, have similar orsuperior LPO inhibition activity to the reference compounds, Trolox(vitamin E derivative, used as a reference material in numerous in vitroand in vivo antioxidant test) and Ebselen (the most promisingantioxidant currently and is in clinical phase III).

Example 9 Protection of Neuron Cells by the Compounds of the InventionExample 9-1 The Culture of Neuron Cells of Cerebral Cortex

[0150] Mixed cortical cell cultures, containing both neuronal and glialelements, were prepared from fetal ICR (Institute Cancer Research) miceat 14-15 days of gestation. Briefly, dissociated cortical cells wereplated onto previously established glial monolayer culture at 2.5hemispheres per 24-multiwell plate (Nunc, USA). The plating mediumconsisted of Eagle's minimal essential medium (Earle's salts, suppliedglutamine-free) supplemented with glucose (final concentration, 20 mM),2 mM glutamine, 5% fetal bovine serum, and 5% horse serum. Ten mMcytosine arabinoside was added to the medium 5-6 days after the platingto halt the growth of non-neuronal cells. Cultures were maintained at37° C. in a humidified CO₂ incubator (5%) and used for experiments afterbetween 10-14 days in vitro (DIV).

[0151] The glial feeder cultures were prepared from neocortices ofpostnatal (1-3 day-old) mice. Dissociated cortical cells were plated at0.25 hemispheres per 24-multiwell plate, in plating medium supplementedwith 5% fetal bovine serum, and 10% horse serum. With this method, mostneurons do not survive, but astrocytes do, resulting in astrocyte-richcultures. Glial cultures were grown to confluency for 10-30 days, whenthey were used to generate mixed cortical cultures.

Example 9-2 Protection of Cortical Neuronal Cell Death Induced by Fe²⁺Ion and Zn²⁺ Ion

[0152] When ferrous iron is placed in normoxic solution, it autooxidizesto produce ROS in the form of hydroxyl radicals, superoxide anion freeradicals, and hydrogen peroxide.

[0153] Cortical cell cultures prepared in Example 9-1 were exposed for24 hours to 30 μM FeCl₂ (Fe) or 35 μM ZnCl₂ (Zn, neuro-excitotoxicfactor), to induce neuronal cell death. Twenty four hour exposure totoxin with or without test compounds was done in serum free Eagle'sminimal essential medium (MEM) supplemented with 20 mM glucose and 38 mMsodium bicarbonate in 5% CO₂ incubator at 37° C. All of compounds weredissolved in DMSO at high concentrations, and then diluted to finalconcentrations in the exposure medium at the time of addition.

[0154] Methods of measuring cell death were as follows:

[0155] Overall cell injury was first estimated in all experiments byexamination of cultures under phase-contrast microscope. Themorphological assessments were usually performed one day after exposureto toxins, at which point the process of cell death was largelycompleted.

[0156] In addition, overall neuronal cell injury was quantitativelyestimated by measuring the activity of lactate dehydrogenase (LDH),released by damaged or destroyed cells, into the extracellular fluid. Asmall amount of LDH was always present in the media of cultures thatunderwent the same exposure procedures but without the addition oftoxins (sham wash controls). This background amount, determined onsister sham wash controls within each experiment, was subtracted fromvalues obtained in toxin-treated cultures. The absolute value of the LDHefflux produced by toxin exposure was quite consistent within sistercultures of single plating, but varied somewhat in cultures of differentplatings. This variability is largely a function of resultant neuronaldensity (which varied despite constant original plating densities,presumably reflecting small variations in cell preparation or serumcharacteristics). Therefore, each LDH value was scaled to the maximalneuronal LDH release (=100) after 24 hours exposure to 30 μM FeCl₂ (Fe)or 35 μM ZnCl₂ (Zn), in sister cultures, where near complete neuronaldeath with no glial damage occurs. Numbers greater than 100 usuallyindicate additional astroglial cell injury.

[0157]FIG. 1-a is a graph showing the results of combined treatment ofTrolox and Fe²⁺ toxin.

[0158]FIG. 1-b is a graph showing the results of combined treatment ofTrolox and Zn²⁺ toxin.

[0159]FIG. 2-a is a graph showing the results of combined treatment ofcompound obtained in Example 1 and Fe²⁺ toxin.

[0160]FIG. 2-b is a graph showing the results of combined treatment ofcompound obtained in Example 1 and Zn²⁺ toxin.

[0161]FIG. 3-a is a graph showing the results of combined treatment ofcompound obtained in Example 2 and Fe²⁺ toxin.

[0162]FIG. 3-b is a graph showing the results of combined treatment ofcompound obtained in Example 2 and Zn²⁺ toxin.

[0163]FIG. 4-a is a graph showing the results of combined treatment ofcompound obtained in Example 3 and Fe²⁺ toxin.

[0164]FIG. 4-b is a graph showing the results of combined treatment ofcompound obtained in Example 3 and Zn²⁺ toxin.

[0165]FIG. 5-a is a graph showing the results of combined treatment ofcompound obtained in Example 4 and Fe²⁺ toxin.

[0166]FIG. 5-b is a graph showing the results of combined treatment ofcompound obtained in Example 4 and Zn²⁺ toxin.

[0167]FIG. 6-a is a graph showing the results of combined treatment ofcompound obtained in Example 5 and Fe²⁺ toxin.

[0168]FIG. 6-b is a graph showing the results of combined treatment ofcompound obtained in Example 5 and Zn²⁺ toxin.

[0169]FIG. 7-a is a graph showing the results of combined treatment ofcompound obtained in Example 6 and Fe²⁺ toxin.

[0170]FIG. 7-b is a graph showing the results of combined treatment ofcompound obtained in Example 6 and Zn²⁺ toxin.

[0171]FIG. 8-a is a graph showing the results of combined treatment ofcompound obtained in Example 7 and Fe²⁺ toxin.

[0172]FIG. 8-b is a graph showing the results of combined treatment ofcompound obtained in Example 7 and Zn²⁺ toxin.

[0173] As seen from FIGS. 1-a to 8-b, the compounds of the presentinvention effectively protected the neuron cell injury induced by Fe²⁺or Zn²⁺ toxin. Therefore, the compounds of the invention can be used asneuroprotective agents in order to protect cerebral neuron cells fromROS and/or excess Zn ions (neuro-excitotoxic factor).

Example 10 Toxicity of the Compounds on the Neuron Cells

[0174] The viability of cortical cell prepared in Example 9-1 wasquantified by lactate dehydrogenase (LDH) assay after exposure for 24hours to the different concentrations of the test compound. Twenty fourhours exposure to the compound was done in serum free Eagle's minimalessential medium (MEM) supplemented with 20 mM glucose and 38 mM sodiumbicarbonate in 5% CO₂ incubator at 37° C. All of compounds weredissolved in DMSO at high concentrations, and then diluted to finalconcentrations in the exposure medium at the time of addition.

[0175] Measurement of cell death was the same as the method in theExample 9-2.

[0176]FIG. 9 is a graph showing the level of cell damage as thetreatment concentration of Trolox increases.

[0177]FIG. 10 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 1 increases.

[0178]FIG. 11 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 2 increases.

[0179]FIG. 12 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 3 increases.

[0180]FIG. 13 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 4 increases.

[0181]FIG. 14 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 5 increases.

[0182]FIG. 15 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 6 increases.

[0183]FIG. 16 is a graph showing the level of cell damage as thetreatment concentration of compound obtained in Example 7 increases.

[0184] As seen from FIGS. 9 to 16, the compounds of the presentinvention exhibit low cytotoxicity. Accordingly, the new compounds ofthe present invention can be administered at large doses.

Example 11 Protection of Cell Damage by Ischemia (In Vivo)

[0185] Male Mongolian gerbils (Meriones unguiculatus) weighing 80-88 gwere used in the present study. Each animal was medicated P.O. withvehicle, Ebselen or various test compounds (60 mg/kg in 10% DMSO), after30 minutes ischemic injury, respectively. 20 animals were allotted intoevery group. The animals were placed under general anesthesia with amixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. Amidline ventral incision was made in the neck. Both common carotidarteries were isolated, freed of nerve fibers, and occluded usingnontraumatic aneurysm clips. Complete interruption of blood flow wasconfirmed by observing the central artery in eyeballs usingophthalmoscope. After five minutes of occlusion, the aneurysm clips wereremoved from both common carotid arteries. Restoration of blood flow(reperfusion) was observed directly under the microscope. Sham-operatedcontrols were subjected to the same surgical procedures except thatcommon carotid arteries were not occluded. Body temperature wasmonitored and maintained at 37° C.±0.5° C. during surgery and during theimmediate postoperative period until the animals recovered fully fromanesthesia. At the designated reperfusion time (4 days), operatedanimals and sham animals were killed.

[0186] Animals were perfused transcardially with phosphate-bufferedsaline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4) at 4 days (n=7) after surgery. The brains were removed,and postfixed in the same fixative for 4 hours. The brain tissues werecryoprotected by infiltration with 30% sucrose overnight. Cornoy fixedspecimens were cut into 30 μm sections on a cryostat. The sections weresequentially stained by Cresyl violet dye.

[0187] Images of staining in the hippocampus of each animal werecaptured with an Applescanner. The brightness and contrast of each imagefile were uniformly enhanced by Adobe Photoshop version 2.4.1, followedby analysis using NIH Image 1.59 software. All data obtained from thequantitative data were analyzed using one-way ANOVA to determinestatistical significance. Bonferroni's test was used for post-hoccomparisons. P values below 0.05 or 0.01 were considered statisticallysignificant.

[0188]FIG. 17-a is a graph showing the protection level of cell damagein case of the treatment of the compound of the invention afterischemia.

[0189]FIG. 17-b is a photomicrograph showing the protection level ofcell damage in case of the treatment of the compound of the inventionafter ischemia.

[0190] As the results, the test compound prepared in Example 4 has moreneuroprotective effects against ischemic neuronal degeneration thanthose of Ebselen. The compound synthesized in Example 4 showed that theprotective effect was 91% in pre-treated groups. In theEbselen-pretreated groups, the effect was 69%.

[0191] In conclusion, we suggest that the compound prepared in Example 4may be an effective candidate as a preventive drug against ischemia,together with as a treatment drug against ischemia.

[0192] As described in detail and illustrated above, the inventionprovides novel dialkylhydroxybenzoic acid derivatives containing metalchelating groups, and the use of said novel compounds as therapeuticsfor treating and/or preventing various medical diseases arising from ROSand/or excess Zn ions. The compounds of the invention possess similar orsuperior lipid peroxidation (LPO) inhibition activity to the referencecompounds of Trolox and Ebselen. While showing lower toxicity, they alsoeffectively inhibit the cerebral neuronal cell death caused by ROSand/or zinc ion and show neuroprotective effects against ischemicneuronal degeneration.

[0193] From the foregoing description, various modifications and changesin the compositions and methods of the invention will occur to thoseskilled in the art. All such modifications coming within the scope ofthe appended claims are intended to be included therein.

What is claimed is:
 1. Dialkylhydroxybenzoic acid derivatives containingmetal chelating groups with the following formula (I), andpharmaceutically acceptable salts thereof:

wherein, R₁ denotes hydrogen, or a straight or branched acyl; R₂ denoteshydrogen or alkyl; and R₃ denotes metal chelating groups such as CO₂H,CONH₂, CONR₄R₅ (where, R₄ denotes hydrogen, alkyl or aryl; and R₅denotes heterocyclic unsaturated or saturated radical having 1 to 4heteroatoms of elements nitrogen, oxygen and/or sulfur from the groupcomprising furanyl, oxazolyl, isooxazolyl, thiophenyl, thiazolyl,isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazoyl,pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl,tetrazolyl, it being possible for the heterocyclic radical to besubstituted once or twice, identically or differently, by halogen,C₁₋₂-alkyl, C₁₋₄-alkoxy, C₁₋₄-alkylthio, hydroxy, mercapto,trifluoromethyl, nitro, phenyl, nitrile, carboxy orC₁₋₄-alkoxycarbonyl).
 2. The compounds according to claim 1, wherein R₁is selected from the group consisting of hydrogen, and straight orbranched C₁₋₁₈-alkyl carbonyl; R₂ is selected from the group consistingof hydrogen, and straight or branched C₁₋₈-alkyl; and, R₃ is selectedfrom the group consisting of CO₂H, CONH₂, and amides CONR₄R₅ (where, R₄represents hydrogen, C₁₋₄ alkyl, aralkyl or cycloalkyl; and, R₅ denotesheterocyclic unsaturated or saturated radical having 1 to 4 heteroatomsof elements nitrogen, oxygen and/or sulfur from the group comprisingfuranyl, oxazolyl, isooxazolyl, thiophenyl, thiazolyl, isothiazolyl,pyrrolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazoyl, pyridyl,pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl,it being possible for the heterocyclic radical to be substituted once ortwice, identically or differently, by halogen, C₁₋₂-alkyl, C₁₋₄-alkoxy,C₁₋₄-alkylthio, hydroxy, mercapto, trifluoromethyl, nitro, phenyl,nitrile, carboxy or C₁₋₄-alkoxycarbonyl).
 3. The compounds according toclaim 2, wherein R₁ is selected from the group consisting of hydrogen,acetyl and propionyl; R₂ is selected from the group consisting ofhydrogen, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl,cyclopentyl and cyclohexyl; and, R₃ is selected from the groupconsisting of CO₂H, CONH₂, and amides CONR₄R₅ (where, R₄ representshydrogen, methyl, ethyl, propyl, benzyl, cyclopentyl or cyclohexyl; and,R₅ denotes heterocyclic unsaturated or saturated radical having 1 to 4heteroatoms of elements nitrogen, oxygen and/or sulfur from the groupcomprising oxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl,thiadiazoyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,triazolyl, tetrazolyl, it being possible for the heterocyclic radical tobe substituted once or twice, identically or differently, by fluorine,chlorine, bromine, methyl, methoxy, ethoxy, methylmercapto, hydroxy,mercapto, trifluoromethyl, nitro, phenyl, nitrile, carboxy,methoxycarbonyl, or ethoxycarbonyl).
 4. A pharmaceutical compositionuseful as antioxidant and/or metal chelating agent which comprises as anactive ingredient an effective amount of the compound of formula (I)defined in claim 1, in combination with one or more pharmaceuticallyacceptable carriers or excipients.
 5. A neuroprotective agent comprisingas an active ingredient an effective amount of the compound of formula(I) defined in claim 1, in combination with one or more pharmaceuticallyacceptable carriers or excipients.
 6. The pharmaceutical compositionaccording to claim 4, wherein the carrier is an oral carrier.
 7. Thepharmaceutical composition according to claim 4, wherein the carrier isan injectable carrier.
 8. A method for treating a living body afflictedwith a condition requiring an antioxidant and/or metal chelating agent,which comprises a step of administering to the living body an amount ofthe compound of the formula (I) defined in claim 1 which is effectivefor alleviation of said condition.
 9. A method for treating a livingbody with acute or progressive neurodegenerative disorders, whichcomprises a step of administering to the living body an amount of thecompound of formula (I) defined in claim 1 which is effective foralleviation of said disorders.
 10. The method according to claim 9,wherein the acute or progressive neurodegenerative disorders areselected from the group consisting of stroke, Parkinson's disease andAlzheimer's disease.
 11. The method according to claim 9, wherein theliving body exhibits symptoms of stroke.